Discontinuation of smallpox vaccination since 1971 has led to waning of acquired immunity in the U.S. general population, thus, raising the risk of major epidemics from intentional release of smallpox by terrorists or unfriendly governments. The CDC has shown that vaccinia immunization, which led to the eradication of smallpox worldwide, caused excess rates of severe complications and death among older persons. Since higher morbidity and mortality rates from many other viral infections in older persons have been attributed to a deficiency in virus-specific HLA-restricted CTL response, the elderly will be expected to possess reduced vaccinia-specific CTL activity and therefore be more susceptible to smallpox disease as well as complications from vaccinia immunization. The goals of this study are to: 1) profile the sero-prevalence and memory CD8+ CTL activity status against vaccinia in representative young adult and elderly U.S. populations, 2) identify easily detectable and quantifiable surrogates of CTL competence against smallpox infection and 3) explore immunological basis for complications from vaccinia vaccination. Levels of vaccinia-specific memory CD8 CTL activity will be determined in peripheral blood lymphocytes of U.S. born healthy adults (35-49 and 50-64 years old) and elderly adults (~65 years old) who have previously been vaccinated with vaccinia and compared with young adults (18-32 years old) who are recent (<1 year) vaccinia recipients or vaccinia naive. Virus serum neutralization antibody and virus specific IgG antibody titers will be determined and correlated with CTL activity. The frequency of CD8+ T cells expressing interferon (IFN)-gamma (a CTL surrogate) and interleukin (IL)-4 (a CTL antagonist) will be measured using ELISPOT and flow cytometry. The functions of IFN-gamma and IL-4 will be assessed using antisense oligonucleotides and/or antibodies. Selected pro-inflammatory cytokines and chemokines (IL-1, TNF-a, IL-6, IL-8 and lL-13) will be analyzed. It is postulated that among previously vaccinated persons: a) the elderly will have significantly reduced CD8+ CTL memory in comparison to younger persons, b) levels of vaccinia-specific antibody and memory CD8+ CTL activity will decline with time, c) IFN-gamma production will be a good surrogate of CTL activity and d) increases in specific pro-inflammatory cytokines or chemokines will be associated with the intensity of vaccinia vaccination lesions. The results should provide guidance in decisions to revaccinate the public, particularly the elderly and other immunodeficient persons, in the event of a biological attack with smallpox and offer needed insight into the mechanism of vaccinia complications in the elderly. Data from recent vaccinees might also serve as reference values for new smallpox vaccines that might be developed since specific correlates of immunity against smallpox (other than a vaccine "take") are currently not available. By correlating these responses with vaccinia lesion sizes in new vaccinees, markers of severe complications from vaccinia vaccination may be identified.